SIRT1, an NAD+-dependent enzyme with deacetylase activity, is an important regulator of mammalian fuel utilization pathways and energy homeostasis as well as longevity. SIRT1 activity is upregulated in response to low nutrient availability, such as fasting or caloric restriction, and results in the activation of pathways associated with gluconeogenesis, fatty acid oxidation, mitochondria! proliferation, and antioxidant protection. Despite the clear association between nutrient deprivation and SIRT1 activation, very little is actually known about the mechanisms behind how SIRT1 is able to sense and subsequently respond to changes in nutrient load. Elucidation of these mechanisms is an important first step in the rational design of compounds that are able modulate SIRT1 activity. The long-term objective of this proposal, therefore, is to dissect the regulatory mechanisms surrounding nutrient activation of SIRT1. The first aim of this proposal is to identify endogenous modulators of SIRT1 activity that change in response to nutrient deprivation. The first rational modulator to be tested is NAD+, which is known to increase in vivo in response to fasting. Using cultured cells, NAD+ levels will be altered by overexpression/knockdown of the enzymes involved NAD+ biosynthesis. SIRT1 activity will be assessed by changes in the acetylation state of immunoprecipitated SIRT1 targets. Additional modulators will be screened by a less biased approach by testing the ability of purified fractions of cells grown under either nutrient replete or nutrient deficient conditions to enhance SIRT1 activity in an in vitro deacetylase assay. The second aim is to map domains of the SIRT1 protein that are responsible for enhancement of deacetylase activity under conditions of nutrient deprivation. Truncated mutants of SIRT1 will be cloned and ectopically expressed in SIRT1 (-/-) MEFs cultured under conditions of nutrient sufficiency/deprivation. The acetylation state of SIRT1 targets will be measured to determine what effect sequence deletion had on SIRTI's ability to sense nutrient deprivation. The third aim of this proposal is to evaluate whether amino acids levels modulate SIRT1 activity. Mammalian cell lines will be cultured in medium that is either amino acid replete or deficient in particular amino acids. SIRT targets will then be immunoprecipitated and their acetylation state evaluated. PUBLIC HEALTH RELEVANCE: This proposal seeks to better our understanding of how the body senses nutrients and regulates pathways of energy usage. This is important because three major diseases (obesity, type II diabetes, and cardiovascular disease) all involve dysregulations in how the body uses energy from food. - follow PHS 416-1 instructions)